Directed Evolution of Artificial Aldolase for Enantioselective Biosynthesis of 3-Hydroxy-Oxindoles

Enantioenriched 3-hydroxyoxindole scaffolds are prevalent in numerous natural products and exhibit significant biological activity. However, biosynthetic methods for their production remain relatively limited. Here, we report an enantioselective biosynthesis of 3-hydroxy-oxindoles by a myoglobin-based artificial aldolase. By site-specifically incorporating a synthetic secondary amine cofactor into the apo-myoglobin scaffold, the engineered artificial aldolase catalyzes C-C bond formation between isatins and activated ketones, yielding chiral 3-hydroxy-oxindoles with excellent enantioselectivity (up to 96:4 er) and reactivity (up to 99% yield) after directed evolution. The resulting artificial enzyme combines the non-natural catalytic activity of chemical small-molecule catalysts with the tunability of protein scaffolds, enabling efficient access to chiral oxindole architectures with pharmaceutical relevance.